In the field of medical or biological sciences, there has always been the demand to produce large quantities of microorganisms such as viruses, bacteria, yeasts, other fungi, parasites and prions. The resulting microorganisms can be used to isolate and purify microbial proteins, generate vaccines, or provide infectious microorganisms for laboratory or medical studies. Recently, the new development of virus therapy has further necessitated the need for efficient production of infectious viruses.
Reovirus therapy (U.S. Pat. No. 6,136,307) is an example of virus therapy. Reovirus is a double-stranded RNA virus with a segmented genome. The receptor for the mammalian reovirus is a ubiquitous molecule, therefore reovirus is capable of binding to a multitude of cells. However, most cells are not susceptible to reovirus infection and binding of reovirus to its cellular receptor results in no viral replication or virus particle production. This is probably the reason why reovirus is not known to be associated with any particular disease.
It was discovered recently that cells transformed with the ras oncogene become susceptible to reovirus infection, while their untransformed counterparts are not (Strong et al., 1998). For example, when reovirus-resistant NIH 3T3 cells were transformed with activated Ras or Sos, a protein which activates Ras, reovirus infection was enhanced. Similarly, mouse fibroblasts that are resistant to reovirus infection became susceptible after transfection with the EGF receptor gene or the v-erbB oncogene, both of which activate the ras pathway (Strong et al., 1993; Strong et al., 1996). Thus, reovirus can selectively infect and kill cells with an activated ras pathway.
Ras pathway activation accounts for a large percentage of mammalian tumors. Activating mutations of the ras gene itself occur in about 30% of all human tumors (Bos, 1989), primarily in pancreatic (90%), sporadic colorectal (50%) and lung (40%) carcinomas, as well as myeloid leukemia (30%). Activation of factors upstream or downstream of ras in the ras pathway is also associated with tumors. For example, overexpression of HER2/Neu/ErbB2 or the epidermal growth factor (EGF) receptor is common in breast cancer (25-30%), and overexpression of platelet-derived growth factor (PDGF) receptor or EGF receptor is prevalent in gliomas and glioblastomas (40-50%). EGF receptor and PDGF receptor are both known to activate ras upon binding to their respective ligand, and v-erbB encodes a constitutively activated receptor lacking the extracellular domain. Accordingly, reovirus therapy, which is highly selective for ras-associated tumor cells, can be used to treat a vast variety of tumors.
Reovirus can be produced and purified in bulk preparations (U.S. Patent Application Publication Number 2002/0037576 A1). To ensure that the reovirus preparation does not contain adventitious agents which may result in undesired side effects, the preparation is validated by using a susceptible cell line and anti-reovirus antibodies. Thus, the cell line is exposed to either the virus preparation alone, or the virus preparation that has been neutralized by a reovirus-specific neutralizing antibody. If the antibody neutralized virus preparation is still pathogenic to the cell line, the virus preparation must contain an adventitious virus or other organism. The preparation is then discarded or further purified.
This validating protocol is expensive, as it requires large amounts of high affinity, high titer antibodies to neutralize the virus. This problem is further exacerbated now that we can produce reovirus very efficiently, and the requirement for antibody is even higher. Therefore, a more cost effective approach is desirable.